The long-term objective of this project is to discover new therapeutics for leishmaniasis and Chagas disease. These infectious diseases are caused by parasitic protozoa of the family, Trypanoisomatidae. Leishmaniasis affects 12 million people in 88 countries with an annual incidence of about 2 million people. Visceral leishmaniasis is fatal if untreated and cutaneous leishmaniasis causes serious morbidity, Chagas disease is caused by Trypanosoma cruzi and is endemic in over 20 countries in Latin America. An estimated 16-18 million persons are chronically infected and at risk for developing life threatening cardiomyopathy or megasyndromes of the gastrointestinal tract. The treatments for leishmania infections are inadequate because of the toxicity of currently available drugs and because of the need to administer the drugs by injection. Treatments for Chagas diseases are highly toxic and do not cure most patients with chronic phase disease. Leishmania species and Trypanosoma cruzi synthesize membrane sterols similar to those of fungi. It has been shown that a number of anti-fungal drugs that act on sterol biosynthesis or directly on ergosterol have anti- lishmanial and anti-trypanosomal effects. Our hypothesis is that a characterization of the sterol biosynthesis pathway in Trypanosomatids will lead to novel drug treatments. The specific aims of the research are: 1) Clone Trypanosomatid homologs of selected sterol biosynthesis genes. We will concentrate on five enzymes that are potentially enzymes that are potentially excellent drug targets: squalene synthetase, squalene epoxidase, C14 demethylase, delta 14-reductase, and C8 isomerase. Genes will be cloned with the use of sequences in the parasite genome databases that have high homology scores to sterol biosynthesis genes of other organisms. 2) Characterize the Trypanosomatid homologs of sterol biosynthesis genes. The enzymatic function of the cloned parasite genes will be investigated by heterologous complementation of yeast mutants. We will be prepared for the possibility that Trypanosomatids make sterols by a route that differs from the yeast pathway. 3) Interrupt the sterol biosynthesis genes in Leishmania mexicana and Trypanosoma cruzi. Targeted knockout of sterol biosynthesis genes will be done using selectable drug markers. The enzymes that are shown to be essential for parasite viability will be prioritized for subsequent drug studies. 4) Screen sterol biosynthesis inhibitors for anti-Trypanosomatid activity. Test compounds will be obtained from collaborating investigators and pharmaceutical companies. Drugs will be screened in high throughput in vitro systems. The best compounds will be tested in murine models of Chagas disease and leishmaniasis. The drugs discovered in this research program will hopefully provide better future treatment for patients with these devastating diseases.